SMS 598: Application of Remote and In-situ Ocean Optical Measurements to Ocean Biogeochemistry

1. SMS 598: Application of Remote and In-situ Ocean Optical Measurements to OceanBiogeochemistry Fluorescence Mary Jane Perry6 July 2007

2. 1. What is fluorescence?2. What fluoresces in the ocean?3. Fluorescence as a proxy4. Types of fluorescence5. Instrumentation issues6. Examples7. Today’s labs

3. Fluorescence: Re-emission of energy as a photon as an electron relaxes from a electronic excited stateFraction of energy absorbed atshorter wavelengths (higher frequency, higher energy) is re-emitted as a photon at longer wavelengths (lower frequency, lower energy). E = h = hc/Property of some molecules (not all)

4. Collin’s lecture Tuesday:Absorption of a photon occurs if AND ONLY IFthe energy of the photon (E = h = hc/) is equal to the energy difference of an electron in the ground state (S0) and higher electronic states (Sn).Absorption is an “electronic transition”; (O(10-15 s))

5. Collin’s lecture Tuesday:Absorption of a photon occurs if AND ONLY IFthe energy of the photon (E = h = hc/) is equal to the energy difference of an electron in the ground state (S0) and higher electronic states (Sn).Absorption is an “electronic transition”; (O(10-15 s)) Vibrational states w/in electronic states Primary mechanism of energy loss to permit an electron to relax or return to S0 is by loss of heat (IR radiation); so-called radiationless decay; (O(10-12 s)).

6. http://www.micro.magnet.fsu.edu/primer/java/fluorescence/exciteemit/index.htmlhttp://www.micro.magnet.fsu.edu/primer/java/fluorescence/exciteemit/index.html http://micro.magnet.fsu.edu/primer/java/jablonski/jabintro/index.html

7. Chlorophyll absorption (direct or via accessory pigs) Chlorophyll excited electron: Photochemistry (charge separation) Heat (many pathways) Fluorescence at 686 nm (O(10-9 s))

8. Fluorescence emission1. always from lowest vibrational state of Sn2. red shifted – Stokes shift (higher , lower E)3. mirror image of absorption

9. F = E() . conc .fF = E() . a() .fwhereE() is excitation lamp energy conc is concentration a is () is absorption fis quantum yield of fluorescence = moles photons fluoresced moles photons absorbedif fwere constant, F ~ conc or a

10. Collin, Station 2 of today’s lab fluorescence excitation/emission* match of wavelengths E() and a() * energy transfer of chlorophyll ain vivo (living cell) vs. in vitro (out of cell, solvent)

11. What fluoresces in the ocean?Chlorophyll a– red(note, chlorophyll b only fluoresces in solvent –in vitro– so tightly coupled to chlorophyll a in membrane)PE– phycoerythrin (orange)CDOM – broad excitation, with some peaksGreen fluorescent protein (used in molecular staining) –from coral, jellyfish and some protozoans

12. CDOM F – proxy for aCDOMfor radiative transfer aCDOM– proxy for DOM for carbon cycling PE – specific taxa Chl F – proxy for Chl – proxy for phytoplankton and input to productivity and carbon models

13. Our ability to use proxies in any quantitative sense depends on this relationship:F = E() . conc .ff depends on temperature and environment(pH, ionic strength, interaction with other molecules for dissipation of energy, etc.) chlorophyll a fluorescence in vitro (solvent, acetone) f ~ 0.33 chlorophyll a fluorescence in vivo (living cell) f ~ <0.05 – 0.03

14. Three types of fluorescence:1) active – artificial light source for E()– static: use for profiles of chl fluorescence; moorings; mobile platforms– time resolved (true tr is ~ femo/pico s for chemistry, like whole burning in CDOM; could consider pump & probe, variable F) 2) passive – sun is light source for E()

15. Instrumentation issues (a few):Sensors – trend toward smaller, lighter, low power, robust, more sensitive, smaller sensing volume; biofouling issuesE() varies among instruments aps() will vary among cells, based on accessory pigments; does E() match aps()? Manufacturer change in LEDs to 470 nm. Different (), different accessory pigmentsCalibrationsensor side: dark reading, temperature response of electronics and optics, stability and driftfluorophore side: (CDOM, Chl): temperature response (-1–2%/ºC), behavior of fAttenuation of signal – turbidity (nonlinear response)

16. Example of passive or solar-stimulated fluorescence from Babin and Huot (recall Curt’s lecture, Hydrolight output)

17. Other issues:1) satellite images only available on clear days; bias of high light/quenching; what is f? 2) how to interpret, E(), a (), depth resolution from Babin and Huot;they caution its use in turbid waters (not F)

18. F = E() . conc .f Note: important temperature effect on f (watch out if room temp changes) ~ - 1–2% F / ºC F Not just chl a, also degradation pigments (pheophytin a). Fo reading = chl a + pheo; add H+; Fa reading = new pheo + old pheo (2 readings, 2 equations, 2 unknowns) BUT: also chlorophyll b and its degradation products. Filter set. concentration Example of active /static benchtop application (Ststion 1):fluorescence of solvent-extracted chlorophyll a

19. Chlorophyll mg/m3 PAR GoMOOS Buoy E (Roesler) From Falkowski and Raven 1997 Chlorophyll fluorescence and extracted concentration of chlorophyll early AM vs. noon. Example of active /static in situ application for living cells (Station 3) two types: flush-face and flow-through.Used on ship-based profiling systems, moorings, floats and gliders. Glider fluorescence, Wash. Coast

20. Mid-day fluorescence quenching Example of mid-day fluorescence quenching 0 • Quenching observed to 11m • Fluoresence quenched up to 80% at surface Depth (m) 40 112.48 Year Day 118.2 -- Mixed Layer Depth (MLD) So maybe for biomass, should we concentrate on night-time measurements in vivo fluorescence measurements? Sackmann et al, MS.

21. Mid-day fluorescence quenching • MORNING • MID-DAY • AFTERNOON Sackmann et al., unpub.

22. Mid-day fluorescence quenching • MORNING • MID-DAY • AFTERNOON Sackmann et al., unpub.

23. Mid-day fluorescence quenching • MORNING • MID-DAY • AFTERNOON Sackmann et al., unpub.

24. PAR Fluorescence Time Figure 2: Damariscotta River in situ chlorophyll a fluorescence and PAR (μmol photons/s/m2)vs. time.

25. PAR Fv/Fm Time Figure 4:Normalized variable fluorescence (Fv/Fm) and PAR (μmol photons/s/m2) vs. time.

26. Fluorescence induction curve (issues of timing and of initial state) Fv = Fm - Fo Fluorescence induction curve: rapid rise and slow decline

27. Fluorescence induction curves, for dark-adapted cells Fast rise (< second); #1 – low light; #2 – high light adapted; #3 DCMU Slow rise (< minute) photoreduction of QA to QA- and connectivity among Reaction Centers photochemical, thermal and other quenching

28. Saturday experiment* automoatic: continuous measurement of PAR, F, and bb* student teams; hourly sampling of chlorophyll a (extract) and Fv/Fm with FIRe (Station 4)Questions:How does incident light affect in situ F of phytoplankton in the DRE (tides, mixing, variable PAR)?What is the relationship between quenching or photoinhibition of in situ F and Fv/Fm? And could Fv/Fm help to interpret F?Lightening ––––– don’t sample!